RESUMEN
Objective: To design and characterize aerosol microparticles (MP) to provide sustained release of the water-soluble compound sulforhodamine B (SRB) and achieve effective aerosol dispersion. Significance: Modulating the release of water-soluble compounds remains a challenge in pulmonary drug delivery. Methods: SRB and water made up an aqueous solution, while acetalated dextran (Ac-Dex) and isopropyl alcohol made up an organic solution. The two solutions were mixed together, and the solution was spray dried to produce MP. MP were characterized for morphology, size, release kinetics, aerosol dispersion, and cellular interactions. Results: Ac-Dex MP exhibited corrugated morphology and aerodynamic diameters from 2.06 to 2.86 µm. MP deposited in all stages of a Next Generation Impactor, with >90% fine particle fraction. MP exhibited encapsulation efficiencies >129% with SRB loading values up to 16.7 µg SRB/mg MP. MP exhibited sustained release of SRB at pH 7 and fast release at pH 5. In vitro experiments showed minimal cytotoxicity, successful uptake of MP in epithelial cells, and no disruption to the integrity of epithelial monolayers. Conclusions: Ac-Dex MP systems demonstrated the ability to provide sustained the release of a water-soluble therapeutic in addition to effective aerosol dispersion for pulmonary applications.
Asunto(s)
Aerosoles/química , Preparaciones de Acción Retardada/química , Dextranos/química , Colorantes Fluorescentes/administración & dosificación , Rodaminas/administración & dosificación , Acetilación , Administración por Inhalación , Cristalización , Liberación de Fármacos , Inhaladores de Polvo Seco , Colorantes Fluorescentes/química , Polvos , Rodaminas/química , Agua/químicaRESUMEN
Three-dimensional (3 D) cell culture platforms are increasingly being used in cancer research and drug development since they mimic avascular tumors in vitro. In this study, we focused on the development of a novel air-grown multicellular spheroid (MCS) model to mimic in vivo tumors for understanding lung cancer biology and improvement in the evaluation of aerosol anticancer therapeutics. 3 D MCS were formed using A549 lung adenocarcinoma cells, comprising cellular heterogeneity with respect to different proliferative and metabolic gradients. The growth kinetics, morphology and 3 D structure of air-grown MCS were characterized by brightfield, fluorescent and scanning electron microscopy. MCS demonstrated a significant decrease in growth when the tumor-penetrating peptide iRGD and paclitaxel (PTX) were coadministered as compared with PTX alone. It was also found that when treated with both iRGD and PTX, A549 MCS exhibited an increase in apoptosis and decrease in clonogenic survival capacity in contrast to PTX treatment alone. This study demonstrated that coadministration of iRGD resulted in the improvement of the tumor penetration ability of PTX in an in vitro A549 3 D MCS model. In addition, this is the first time a high-throughput air-grown lung cancer tumor spheroid model has been developed and evaluated.
Asunto(s)
Adenocarcinoma/patología , Apoptosis/efectos de los fármacos , Técnicas de Cultivo de Célula/métodos , Neoplasias Pulmonares/patología , Oligopéptidos/administración & dosificación , Paclitaxel/farmacología , Esferoides Celulares/patología , Adenocarcinoma/tratamiento farmacológico , Antineoplásicos Fitogénicos/farmacología , Proliferación Celular/efectos de los fármacos , Evaluación de Medicamentos , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Esferoides Celulares/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Pulmonary arterial hypertension (PAH) is an incurable cardiovascular disease characterized by high blood pressure in the arteries leading from the heart to the lungs. Over two million people in the USA are diagnosed with PAH annually and the typical survival rate is only 3 years after diagnosis. Current treatments are insufficient because of limited bioavailability, toxicity, and costs associated with approved therapeutics. Aerosol delivery of drugs is an attractive approach to treat respiratory diseases because it increases localized drug concentration while reducing systemic side effects. In this study, we developed phospholipid-based aerosol microparticles via spray drying consisting of the drug tacrolimus and the excipients dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol. The phospholipid-based spray-dried aerosol microparticles were shown to be smooth and spherical in size, ranging from 1 to 3 µm in diameter. The microparticles exhibited thermal stability and were amorphous after spray drying. Water content in the microparticles was under 10%, which will allow successful aerosol dispersion and long-term storage stability. In vitro aerosol dispersion showed that the microparticles could successfully deposit in the deep lung, as they exhibited favorable aerodynamic diameters and high fine particle fractions. In vitro dose-response analysis showed that TAC is nontoxic in the low concentrations that would be delivered to the lungs. Overall, this work shows that tacrolimus-loaded phospholipid-based microparticles can be successfully created with optimal physicochemical and toxicological characteristics.
Asunto(s)
Aerosoles/química , Descubrimiento de Drogas/métodos , Inhaladores de Polvo Seco/tendencias , Microesferas , Fosfolípidos/química , Células A549 , Administración por Inhalación , Aerosoles/administración & dosificación , Aerosoles/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Relación Dosis-Respuesta a Droga , Inhaladores de Polvo Seco/métodos , Excipientes/administración & dosificación , Excipientes/química , Excipientes/metabolismo , Humanos , Hipertensión Pulmonar/tratamiento farmacológico , Hipertensión Pulmonar/metabolismo , Tamaño de la Partícula , Fosfolípidos/administración & dosificación , Fosfolípidos/metabolismo , Resultado del TratamientoRESUMEN
Cooperation between DNA, RNA and protein regulates gene expression and controls differentiation through interactions that connect regions of nucleic acids and protein domains and through the assembly of biomolecular condensates. Here, we report that endoderm differentiation is regulated by the interaction between the long non-coding RNA (lncRNA) DIGIT and the bromodomain and extraterminal domain protein BRD3. BRD3 forms phase-separated condensates of which the formation is promoted by DIGIT, occupies enhancers of endoderm transcription factors and is required for endoderm differentiation. BRD3 binds to histone H3 acetylated at lysine 18 (H3K18ac) in vitro and co-occupies the genome with H3K18ac. DIGIT is also enriched in regions of H3K18ac, and the depletion of DIGIT results in decreased recruitment of BRD3 to these regions. Our findings show that cooperation between DIGIT and BRD3 at regions of H3K18ac regulates the transcription factors that drive endoderm differentiation and suggest that protein-lncRNA phase-separated condensates have a broader role as regulators of transcription.
Asunto(s)
Diferenciación Celular , Endodermo/citología , Histonas/metabolismo , Células Madre Embrionarias Humanas/citología , Transición de Fase , ARN Largo no Codificante/genética , Factores de Transcripción/metabolismo , Acetilación , Endodermo/metabolismo , Genoma Humano , Histonas/genética , Células Madre Embrionarias Humanas/metabolismo , Humanos , Lisina/genética , Lisina/metabolismo , Dominios Proteicos , Procesamiento Proteico-Postraduccional , Factores de Transcripción/genéticaRESUMEN
The publisher regrets that an author was not mentioned in the chapter by mistake. The details of the author are provided below:Archna Dhasmana - Department of Polymer and Process Engineering, Indian Institute of Technology, Roorkee, India.
RESUMEN
Decellularization is the process of removal of native cells from tissue, leaving behind a three-dimensional (3D) ultrastructure of extracellular matrix (ECM) proteins while preserving the bioactivity and mechanics of the tissue. It offers a unique top-down approach for fabricating ECM based natural scaffold for tissue engineering application. Herein, this chapter presents the fabrication of decellularized scaffold employing different methods: whole organ perfusion, immersion and agitation, pressure gradient, and supercritical fluid. The decellularized scaffold aims to exploit the nature-designed 3D architecture, a successful platform technology, for creating scaffolding materials for tissue engineering and regenerative medicine.
Asunto(s)
Matriz Extracelular/química , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Animales , Cromatografía con Fluido Supercrítico/métodos , Matriz Extracelular/ultraestructura , Congelación , Humanos , Perfusión/métodos , Presión , Medicina Regenerativa/métodos , Solventes/químicaRESUMEN
Biocompatible, biodegradable polymers are commonly used as excipients to improve the drug delivery properties of aerosol formulations, in which acetalated dextran (Ac-Dex) exhibits promising potential as a polymer in various therapeutic applications. Despite this promise, there is no comprehensive study on the use of Ac-Dex as an excipient for dry powder aerosol formulations. In this study, we developed and characterized pulmonary drug delivery aerosol microparticle systems based on spray-dried Ac-Dex with capabilities of (1) delivering therapeutics to the deep lung, (2) targeting the particles to a desired location within the lungs, and (3) releasing the therapeutics in a controlled fashion. Two types of Ac-Dex, with either rapid or slow degradation rates, were synthesized. Nanocomposite microparticle (nCmP) and microparticle (MP) systems were successfully formulated using both kinds of Ac-Dex as excipients and curcumin as a model drug. The resulting MP were collapsed spheres approximately 1µm in diameter, while the nCmP were similar in size with wrinkled surfaces, and these systems dissociated into 200nm nanoparticles upon reconstitution in water. The drug release rates of the Ac-Dex particles were tuned by modifying the particle size and ratio of fast to slow degrading Ac-Dex. The pH of the environment was also a significant factor that influenced the drug release rate. All nCmP and MP systems exhibited desirable aerodynamic diameters that are suitable for deep lung delivery (e.g. below 5µm). Overall, the engineered Ac-Dex aerosol particle systems have the potential to provide targeted and effective delivery of therapeutics into the deep lung.
Asunto(s)
Administración por Inhalación , Aerosoles/química , Curcumina/administración & dosificación , Dextranos/química , Liberación de Fármacos , Excipientes/química , Pulmón/efectos de los fármacos , Tamaño de la Partícula , PolvosRESUMEN
In the present study, goat-lung scaffold was fabricated by decellularization of lung tissue and verified for complete cell removal by DNA quantification, DAPI and H&E staining. The scaffold was then modified by crosslinking with quercetin and nanohydroxyapatite (nHAp), and characterized to evaluate the suitability of quercetin-crosslinked nHAp-modified scaffold for regeneration of bone tissue. The crosslinking chemistry between quercetin and decellularized scaffold was established theoretically by AutoDock Vina program (in silico docking study), which predicted multiple intermolecular hydrogen bonding interactions between quercetin and decellularized scaffold, and FTIR spectroscopy analysis also proved the same. From MTT assay and SEM studies, it was found that the quercetin-crosslinked nHAp-modified decellularized scaffold encouraged better growth and proliferation of bone-marrow derived mesenchymal stem cells (BMMSCs) in comparison to unmodified decellularized scaffold, quercetin-crosslinked decellularized scaffold and nHAp-modified decellularized scaffold. Alkaline Phosphatase (ALP) assay results showed highest expression of ALP over quercetin-crosslinked nHAp-modified scaffold among all the tested scaffolds (unmodified decellularized scaffold, quercetin-crosslinked decellularized scaffold and nHAp-modified decellularized scaffold) indicating that quercetin and nHAp is very much efficient in stimulating the differentiation of BMMSCs into osteoblast cells. Alizarin red test quantified in vitro mineralization (calcium deposits), and increased expression of alizarin red over quercetin-crosslinked nHAp-modified scaffold indicating better stimulation of osteogenesis in BMMSCs. The above findings suggest that quercetin-crosslinked nHAp-modified decellularized goat-lung scaffold provides biomimetic bone-like microenvironment for BMMSCs to differentiate into osteoblast and could be applied as a potential promising biomaterial for bone regeneration.
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Células de la Médula Ósea/metabolismo , Regeneración Ósea , Durapatita/química , Pulmón/química , Células Madre Mesenquimatosas/metabolismo , Nanopartículas/química , Quercetina/química , Andamios del Tejido/química , Animales , Células de la Médula Ósea/citología , Cabras , Humanos , Células Madre Mesenquimatosas/citologíaRESUMEN
Tacrolimus (TAC) has exhibited promising therapeutic potential in the treatment of pulmonary arterial hypertension (PAH); however, its application is prevented by its poor solubility, instability, poor bioavailability, and negative systemic side effects. To overcome the obstacles of using TAC for the treatment of PAH, we developed nanocomposite microparticles (nCmP) for the pulmonary delivery of tacrolimus in the form of dry powder aerosols. These particles can provide targeted pulmonary delivery, improved solubility of tacrolimus, the potential of penetration through mucus barrier, and controlled drug release. In this system, tacrolimus-loaded polymeric nanoparticles (NP) were prepared via emulsion solvent evaporation and nCmP were prepared by spray drying these NP with mannitol. The NP were approximately 200nm in diameter with narrow size distribution both before loading into and after redispersion from nCmP. The NP exhibited smooth, spherical morphology and the nCmP were raisin-like spheres. High encapsulation efficacy was achieved both in the encapsulation of tacrolimus in NP and that of NP in nCmP. nCmP exhibited desirable aerosol dispersion properties, allowing them to deposit into the deep lung regions for effective drug delivery. A549 cells were used as in vitro models to demonstrate the non-cytotoxicity of TAC nCmP. Overall, the designed nCmP have the potential to aid in the delivery of tacrolimus for the treatment of PAH.
Asunto(s)
Hipertensión Pulmonar/tratamiento farmacológico , Nanocompuestos/administración & dosificación , Nanocompuestos/química , Tacrolimus/administración & dosificación , Tacrolimus/uso terapéutico , Administración por Inhalación , Aerosoles , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Preparaciones de Acción Retardada/química , Liberación de Fármacos , Humanos , Nanocompuestos/ultraestructura , Tamaño de la Partícula , Polvos , Solubilidad , Tacrolimus/farmacocinética , Tacrolimus/farmacologíaRESUMEN
Decellularized goat-lung scaffold was fabricated by removing cells from cadaver goat-lung tissue, and the scaffold was modified with chitosan/nanohydroxyapatite composite for the purpose of bone tissue engineering applications. MTT assay with osteoblasts, seeded over the chitosan/nanohydroxyapatite-modified decellularized scaffold, demonstrated significantly higher cell growth as compared to the decellularized scaffold without modification. SEM analysis of cell-seeded scaffold, after incubation for 7 days, represented a good cell adhesion, and the cells spread over the chitosan/nanohydroxyapatite-modified decellularized scaffold. Expression of bone-tissue-specific osteocalcin gene in the osteoblast cells grown over the chitosan/nanohydroxyapatite-modified decellularized scaffold clearly signifies that the cells maintained their osteoblastic phenotype with the chitosan/nanohydroxyapatite-modified decellularized scaffold. Therefore, it can be concluded that the decellularized goat-lung scaffold-modified with chitosan/nanohydroxyapatite composite, may provide enhanced osteogenic potential when used as a scaffold for bone tissue engineering.
Asunto(s)
Huesos , Quitosano/química , Durapatita/química , Ingeniería de Tejidos , Animales , Trasplante Óseo , Adhesión Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quitosano/administración & dosificación , Durapatita/administración & dosificación , Cabras/crecimiento & desarrollo , Cabras/fisiología , Pulmón/citología , Nanopartículas/administración & dosificación , Nanopartículas/química , Osteogénesis/efectos de los fármacos , Andamios del Tejido/químicaRESUMEN
The present study aims to fabricate scaffold from cadaver goat-lung tissue and evaluate it for skin tissue engineering applications. Decellularized goat-lung scaffold was fabricated by removing cells from cadaver goat-lung tissue enzymatically, to have cell-free 3D-architecture of natural extracellular matrix. DNA quantification assay and Hematoxylin and eosin staining confirmed the absence of cellular material in the decellularized lung-tissue. SEM analysis of decellularized scaffold shows the intrinsic porous structure of lung tissue with well-preserved pore-to-pore interconnectivity. FTIR analysis confirmed non-denaturation and well maintainance of collagenous protein structure of decellularized scaffold. MTT assay, SEM analysis and H&E staining of human skin-derived Mesenchymal Stem cell, seeded over the decellularized scaffold, confirms stem cell attachment, viability, biocompatibility and proliferation over the decellularized scaffold. Expression of Keratin18 gene, along with CD105, CD73 and CD44, by human skin-derived Mesenchymal Stem cells over decellularized scaffold signifies that the cells are viable, proliferating and migrating, and have maintained their critical cellular functions in the presence of scaffold. Thus, overall study proves the applicability of the goat-lung tissue derived decellularized scaffold for skin tissue engineering applications.